KR20100127824A - Method of denaturing protein with enzymes - Google Patents

Abstract

Provides a method of modifying a protein by applying both protein glutaminase and transglutaminase to a protein, providing a food containing a protein modified by both enzymes, and for modifying a protein containing both enzymes An enzyme preparation is provided, and a method is provided for discovering an appropriate ratio of addition of protein glutaminase and transglutaminase to a protein. The protein glutaminase is added to the protein when the protein glutaminase is added to the protein substantially before or when the protein glutaminase is applied to the protein, or the protein glutaminase is applied to the protein. The protein is modified by setting the addition ratio of and transglutaminase to a predetermined ratio. The appropriate addition ratio of the protein glutaminase and the transglutaminase is determined by the NMR signal intensity of the protein labeled with the isotopically labeled ammonium.

Description

Method of denaturing protein with enzymes

[Description of Related Application]

The present invention is based on the priority claims of Japanese Patent Application No. 2008-066765 (filed March 14, 2008) and Japanese Patent Application No. 2008-294890 (filed November 18, 2008). The content of description shall be introduce | transduced and described in this book, citing.

The present invention relates to a method for modifying a protein, characterized by performing two enzymatic reactions by acting both a transglutaminase and a protein glutaminase, an enzyme that modifies glutamine residues in a protein. In addition, the present invention is a protein containing a protein modified using this method, an enzyme preparation for protein modification and protein glutaminase containing both protein glutaminase and transglutaminase at a specific ratio. The present invention also relates to a method of discovering a proper addition ratio of a transglutaminase to a matrix protein.

Transglutaminase (hereinafter sometimes abbreviated as TG) is an enzyme that catalyzes an acyl transfer reaction between the γ-carboxyamide group and the primary amine of glutamine residues in proteins and peptides. When TG acts as an acyl acceptor on the ε-amino group of the lysine residue in the protein, a crosslinking polymerization reaction of the protein occurs. In addition, when no primary amine is present, water functions as an acyl acceptor and catalyzes a deamide reaction that converts glutamine residues into glutamic acid residues. In application to food, most of them are due to the crosslinking reaction of the protein, and at present, using the properties such as providing or improving the gel formation of the protein, gelling and adhesion of the protein in unheated, fish jelly, ham, sausage, TG formulations have been developed for different uses, such as noodles, tofu and bread.

On the other hand, protein glutaminase (hereinafter sometimes abbreviated as PG) is an enzyme having a function of deamidating an amide group of a glutamine residue in a protein. The detailed description is as disclosed in Patent Document 1, Patent Document 2, Non-Patent Document 1 and the like. According to them, PG acts directly on the amide group in the protein, whereby the glutamine residues in the protein are converted to glutamic acid residues, resulting in the formation of carboxyl groups. Increases and so on. As a result, various functional characteristics such as the solubility of the protein, the increase in water dispersibility, the emulsifying power, and the improvement of the emulsion stability are brought about, and the use of the protein is expected to be expanded.

As described above, it is known that neither TG nor PG are industrially useful enzymes. However, few specific examples have been reported regarding attempts to obtain new added values by using such enzymes in combination. Rather, Patent Literature 1 shows an example of the use of PG as a TG reaction control agent, and in Non-Patent Literature 2, when PG and TG coexist, the crosslinking reaction by TG does not proceed. In addition, the prior knowledge regarding the difference in reactivity between TG and PG is described in more detail. In both enzymes, the target in the substrate protein is the same glutamine residue. However, the substrate specificity for individual glutamine residues in the protein is wider on the PG side than on the TG. It is thought that this is because, in the case of TG, the ε-amino group of the lysine residue is required in addition to the glutamine residue, whereas in the case of PG, only water that is abundantly present in the reaction environment is required in addition to the glutamine residue. For example, the reactivity with respect to glutamine residues in casein shows that the PG has a predominantly higher catalytic efficiency than the TG even in the Km value and the kcat value. Glutamine residues are no longer substrates of TG and no crosslinking reaction proceeds. In fact, the experiment of adding PG during the crosslinking polymerization reaction of casein by TG has shown that the polymerization of casein stops simultaneously with addition. It has also been confirmed that casein, which has been sufficiently deamidated in advance by PG, no longer becomes a substrate of TG, that is, it is not crosslinked polymerized (see Non-Patent Document 2). In addition, Y. S. Gu et al. Investigated the reactivity of the enzyme to α-lactalbumin and reported that 4 out of 6 glutamine residues were deamidated (see Non-Patent Document 3). On the other hand, since actinomycetes TG acts only on glutamine 54, which is one of four, it is described therein that the substrate specificity of PG is wider than that of TG. In addition, Patent Document 1 discloses that TG reaction can be stopped at an appropriate time point by using high reactivity to glutamine residues of PG, and PG is generally known as TG, such as EDTA or ammonium chloride, which is undesirable for addition to food. The possibility of replacing inhibitors is being discussed.

However, no examples have been proposed so far that a combination method of both has been proposed from the viewpoint of stopping the TG reaction by PG. In addition, the conditions under which both TG and PG act on the substrate protein to obtain a desired effect, more specifically, the TG reaction and its modifying effects even under conditions in which the TG reaction is not stopped by PG and coexists with PG. As for the condition that is not damaged, it is not known only by the conventional knowledge. In addition, there are no examples of specific methods for the effective use of both enzymes, such as what effect occurs when the ratio of TG and PG is acted on various substrate proteins, and thus is a completely new attempt. In the case of developing protein products modified by TG and PG, the current situation is to find the optimum reaction conditions for each use.

In addition, since the reaction amount and the degree of the effect vary depending on various factors such as processing time, temperature, enzyme addition amount, substrate type, state, and substrate specificity, the enzyme reaction includes two enzymes having a common substrate. It is not easy to use skillfully, and developers and processors require enormous time and labor to determine the desired food manufacturing conditions for each use. Because the enzyme reaction is different depending on various factors such as processing time, temperature, enzyme addition amount, substrate type, state, and substrate specificity, the reaction amount and degree of effect are different. This is because all combinations need to be reviewed. Therefore, there is a need for the development of a method of skillfully using two enzymes having a common substrate.

By the way, the method of using NMR is known so far as a method of examining the substrate specificity of TG (patent document 3). That is, the reaction of TG is observed by observing by NMR that ¹⁵ N-labeled carboxyamide nitrogen of the glutamine residue which becomes a substrate of TG by interacting arbitrary protein with TG in presence of ^15- N-labeled ammonium chloride. Using this method, protein substrates can be used to simultaneously interpret reactivity and substrate specificity. However, Patent Document 3 does not describe that the carboxyamide nitrogen of the glutamine residue to which PG reacts is ¹⁵ N-labeled.

Patent Document 1: Japanese Unexamined Patent Publication No. 2000-50887

Patent Document 2: Japanese Patent Application Laid-Open No. 2001-218590

Patent Document 3: Japanese Unexamined Patent Publication No. 2002-332295

[Non-Patent Document 1] Yamaguchi et al., Eur. J. Biochem. Vol. 268, 2001, pages 1410-1412

[Non-Patent Document 2] The Latest Technology and Application of Food Enzyme Chemistry-The Prospect of Food Proteomics, (published by CMC), pp. 146-153

Non-Patent Document 3: Y. S. Gu et al., J. Agric. Food Chem. Vol. 49, 2001, pages 5999-6005

The entire disclosures of Patent Documents 1 to 3 and Non-Patent Documents 1 to 3 described above are incorporated by reference in this document.

The following analysis is given in view of the present invention.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method of modifying a protein by acting both a protein glutaminase and a transglutaminase on a protein, to provide a food containing a protein modified by both enzymes, It is to provide an enzyme preparation for protein modification containing both enzymes, and to provide a method for easily finding an appropriate addition ratio of protein glutaminase and transglutaminase.

As a result of intensive studies in view of the above, the inventors of the present invention have considered that TG does not work when PG is present, and it is thought that the combined effect of PG and TG is difficult to be obtained. Even when PG and TG coexisted, it was found that both enzymes act on the substrate protein, thereby obtaining a combined effect of both enzymes. It was also found that PG, like TG, catalyzes the exchange reaction with ammonium salts in addition to catalyzing the deamidation of glutamine residues in proteins. That is, the carboxamide nitrogen of the TG or PG is a glutamine residue for reaction, is ¹⁵ N-labeled. Here, the measurement of detecting the ¹⁵ N label, for example, ¹ H- ¹⁵ N HSQC measurement, revealed that the ¹⁵ N label rate was approximated from the NMR signal intensity, indicating the difference in the respective reactivity. In addition, it was found that the ratio of TG and PG in relation to various proteins correlated well with the ratio of NMR signal intensities. In addition, the PG / TG enzyme weight ratio or activity ratio that gives a range of PG signal intensity ratio (hereinafter referred to as PG / TG signal intensity ratio) to TG in the range of 0.2 to 3.0 does not stop the TG reaction by PG. It has been found that the TG reaction and its modifying effects are not impaired even under conditions in which PG coexists. In other words, it was found that the conditions under which the combined effect of both enzymes can be easily found by applying TG or PG to the substrate in the presence of ¹⁵ N-labeled ammonium and comparing the substrate specificities of both by using NMR. We have succeeded in figuring out the benefits of operating PG and TG simultaneously and then figuring out how to determine the conditions realistically and easily. That is, this invention is as follows.

(1) A method of modifying a protein by applying a protein glutaminase and a transglutaminase to a protein, wherein the time of addition of the protein glutaminase to the protein is substantially different from the time of addition of the transglutaminase. A method of modifying a protein which is concurrent with or before transglutaminase acts on the protein.

(2) The amount of the protein glutaminase and the transglutaminase added to the protein according to item (1), in terms of the activity ratio, of protein glutaminase: transglutaminase = 0.05 to 3 : 1 person method.

(3) The amount of the protein glutaminase and the transglutaminase added to the protein according to item (1) or (2), in terms of weight ratio, of protein glutaminase: transglutaminase = 0.01 to 0.7: 1.

(4) The protein glutamin agent according to any one of the above items (1) to (3), wherein the amount of the protein glutaminase and the transglutaminase to be added to the protein is in the NMR signal intensity ratio. : The modification method which is conditions which become transglutaminase = 0.2-3.0: 1.

(5) The method according to any one of items (1) to (4), wherein the protein is one or two or more selected from milk protein, whey protein, soy protein, wheat gluten, plasma, muscle protein, collagen and gelatin. .

(6) A food containing a protein modified by the method according to any one of items (1) to (5).

(7) Transglutaminase: Protein glutaminase The ratio of protein glutaminase: protein glutaminase: transglutaminase = 0.05-3: 1 enzyme preparation for protein modification.

(8) A transglutaminase: A protein modification enzyme preparation wherein the ratio of protein glutaminase is protein glutaminase: transglutaminase = 0.01 to 0.7: 1 in weight ratio.

(9) Transglutaminase: The ratio of protein glutaminase is a condition that protein glutaminase: transglutaminase = 0.2 to 3.0: 1 in the NMR signal intensity ratio. Enzyme preparation for protein modification.

(10) A transglutaminase and a protein glutaminase are separately acted on the protein in the presence of an isotopically labeled ammonium salt to homologously label the functional groups of the glutamine residues of the protein and measure the NMR signal intensity to obtain the protein. The method of discovering the appropriate addition ratio of the protein glutaminase and the transglutaminase in the process.

According to the present invention, a modification effect of a protein different from the case of using either an enzyme of TG or PG alone, that is, a combination effect of TG and PG, specifically, the properties of the protein contained in the food, on the food This method can provide a novel method for modifying protein, which can obtain the effect of improving the texture (hardness, smoothness, etc.) of the protein-containing food as a result. It is also possible to provide foods containing proteins with novel features obtained using this modification method. In addition, it is possible to provide a method for easily revealing the conditions (addition conditions) suitable for both the enzymes of TG and PG in the protein to act on the substrate to obtain the above effect.

1 is a ¹ H- ¹⁵ N HSQC spectrum in NMR of α-lactalbumin in the presence of ¹⁵ NH ₄ Cl (Experimental Example 1).

TG used in the present invention is widely used for the production of foods such as gelatin, cheese, yoghurt, tofu, fish jelly, ham, sausages, noodles, etc., and for improving the meat quality of meat. (See Japanese Patent Application Laid-Open No. 64) -27471 (Ref. 1)]. Moreover, it is an enzyme used for various purposes industrially, such as being used for manufacture of the heat-stable microcapsule material, immobilized enzyme carrier, etc. TG catalyzes the acyl transfer reaction of the γ-carboxyamide group of the glutamine residue in the peptide chain of the protein molecule. When TG acts as an acyl acceptor of the ε-amino group of the lysine residue in the protein molecule, an ε- (γ-glutamic acid) -lysine bond is formed in and between the protein molecules. In addition, the content of description of Reference 1 is quoted and introduce | transduced into this document.

Examples of TG include calcium-independent and calcium-dependent ones, and both can be used in the present invention. Examples of the former include those derived from microorganisms such as actinomycetes and Bacillus subtilis (see Japanese Patent Application Laid-Open No. 64-27471 (Reference 2)). Examples of the latter include those derived from morphot soy sauce (Japanese Patent Publication No. 1-50382 (reference 3)), human epidermal keratinocyte TG [Phillips, MA et al. (1990); Proc. Natl. Acad. Sci. USA 87, 9333 (Ref. 4)], human blood coagulation factor XIII [Ichinose, A. et al. (1990) Biochemistry 25, 6900. (Ref. 5)], derived from microorganisms such as fungi, derived from animals such as bovine blood and pig blood, derived from fish such as salmon and red snapper (see Seki Nobuo et al. , Journal of Japanese Fisheries Society, Vol. 56, pp. 125 to 132, 1990 (Ref. 6)]. In addition, those produced by genetic recombination (see Japanese Patent Application Laid-Open No. 1-300889 (Ref. 7), Japanese Patent Application Laid-open No. 6-225775 (Ref. 8), Japan). JP-A-7-23737 (reference 9). Any one can be used in the present invention, and is not limited to the origin and manufacturing method. On the other hand, depending on the nature of the isotopically labeled protein, enzymatic reaction in a solvent containing calcium may be undesirable. Therefore, it is preferable to use calcium-independent TG for such a protein. For example, (MTG) derived from the microorganisms (see Japanese Patent Laid-Open No. 64-27471 (Reference Document 10)) and the like also satisfy this condition, and can be said to be optimal at present. For example, Streptoverticillium griseocarneum ) IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamoneum) IFO 12852, Streptoverticillium mobaraense ) derived from IFO 13819 and the like. Hereinafter, the transglutaminase derived from streptoverticillium mobaraens may be called MTG. In addition, the content of the references 2 to 10 is incorporated herein by reference.

The active unit of TG used for this invention is measured and defined as follows. That is, the reaction is carried out using benzyloxycarbonyl-L-glutamyl glycine (Z-Gln-Gly) and hydroxylamine as a substrate, and the resulting hydroxylsamic acid is converted into an iron complex in the presence of trichloroacetic acid. With the absorbance at 525 nm, the amount is measured. In this way, a calibration curve is prepared from the amount of hydroxamic acid, and the quantity of enzyme which produces 1 micromol of hydroxamate in 1 minute is defined as an active unit and one unit. Details of this measurement method have already been reported (see Japanese Patent Application Laid-Open No. 64-27471 (Ref. 11) and the like). In addition, the content of the reference 11 is incorporated by reference in this document.

PG used in the present invention has a function of directly acting on the amide group of the protein to deamid without accompanying cleavage of the peptide bond and crosslinking of the protein. As long as it has the said effect | action, the kind is not specifically limited. Examples of such enzymes include enzymes disclosed in Patent Document 1 and Patent Document 2, but are not particularly limited thereto. PG prepared from the culture solution of the microorganism which produces PG can be used. Although the microorganism used for preparation of PG is not specifically limited, Microorganisms of the genus Creebacterium, Flavobacterium, and Enfedobacter are illustrated.

As a method for preparing PG from the culture medium of microorganisms, known protein separation and purification methods (centrifugal separation, UF concentration, various chromatography using salting out, ion exchange resin and the like) can be used. For example, the culture medium may be centrifuged to remove the cells, and then, salts, chromatography, or the like may be combined to obtain a target enzyme. When the enzyme is recovered from the cells, the desired enzyme can be obtained by, for example, crushing the cells by pressure treatment, ultrasonic treatment, or the like, and then separating and purifying the cells as described above. In addition, after collect | recovering a microbe from a culture liquid previously by filtration, centrifugal processing, etc., you may perform the said series of processes (cell crushing, separation, purification). The enzyme may be powdered by a drying method such as freeze drying or reduced pressure drying, and an appropriate excipient or drying aid may be used at that time.

The active unit of PG of this invention is measured by the method of improving the method of patent document 1, ie, the following method.

(1) 10 µl of an aqueous solution containing PG was added to 100 µl of 176 mM phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and incubated at 37 ° C for 10 minutes, followed by 100 µl of 12% TCA solution. Addition to stop the reaction. At this time, the enzyme concentration is appropriately diluted with 20 mM phosphate buffer (pH 6.0) so that the enzyme concentration is 0.05 mg / ml.

(2) After centrifugation (12000 rpm, 4 ° C, 5 minutes), the supernatant was quantified with NH ₃ by F-kit ammonia (manufactured by Roche). The method is as follows.

(3) To 100 µl of reagent II solution (F-kit accessory), 10 µl of supernatant and 190 µl of 0.1 M triethanolamine buffer (pH 8.0) were added. Measure To the remaining 200 µl, 1.0 µl of Reagent III (glutamate dehydrogenase) was added, and after standing at room temperature for another 20 minutes, the absorbance (E2) of the remaining 200 µl of 340 nm was measured. The ammonia concentration in the reaction solution is determined from a calibration curve showing the relationship between the ammonia concentration prepared using the ammonia standard solution attached to the F-kit and the amount of change in absorbance (340 nm).

(4) The protein concentration is measured at a detection wavelength of 595 nm using a protein assay CBB (Coomassie Brilliant Blue) solution (manufactured by Nakaray Tesque). As a standard, BSA (manufactured by Pierce Corporation) is used.

(5) The activity of PG was calculated | required by the following formula.

In the present invention, the timing of adding PG and TG to the protein to be modified is that the timing of adding PG to the protein is substantially the same as the timing of adding TG, or before TG acts on the protein. Therefore, the method of stopping the TG reaction described in Patent Document 1 by PG addition is not included in the present invention because the timing of addition of PG to the protein is after TG acts on the protein. On the other hand, when the addition time of PG and TG to a protein is substantially simultaneous, it means that PG and TG are thrown in a series of raw material addition processes. Namely, in the production of commercial scale, after the input of the raw material A, the raw material B, the raw material C... It is common to add a plurality of raw materials in order without mixing them in advance, in order to add them in order. However, as long as PG and TG are introduced in a series of raw material addition steps, the "transglutaminase agent" The timing of addition of the protein glutaminase to the protein is substantially simultaneous ”. Of course, it is needless to say that even if a plurality of raw materials including PG and TG are mixed in advance and added, they are included in the "substantial simultaneous" of the present invention.

The temperature at which PG and TG are reacted is generally about 3 to 60 ° C., and when maintained at this temperature, the reaction of both proceeds in about 1 minute to about 48 hours. However, it is preferable to carry out about 5 minutes to about 24 hours at about 5-50 degreeC preferably. The addition method of PG and TG to foodstuffs is good only if PG and TG are added to the raw material containing the protein used as a substrate, or it is added together with another raw material. The amount of PG and TG added can be changed freely depending on the type of protein to be modified, the final product or the desired effect. For example, the amount of TG added is 0.1 to 100 per gram of protein in the food raw material. Unit. On the other hand, PG is good to add 0.01-120 units normally per gram weight of protein in a food raw material. However, the said addition amount of both is a reference | standard, and is not necessarily limited to these as long as the desired effect of this invention is shown.

In the present invention, the ratio of PG and TG acting on the protein is important. When the addition time of the PG to the protein is substantially the same as the addition time of the TG, or before the TG acts on the protein, the amount of the protein glutase and the transglutaminase added to the protein is in terms of the activity ratio. If PG: TG = 0.05-3: 1, preferably PG: TG = 0.05-2: 1, since both PG and TG act on the protein, the protein can be modified. Or PG: TG = 0.01 to 0.7: 1, preferably PG: TG = 0.01 to 0.4: 1, more preferably PG: TG = 0.01 to 0.3: 1 in weight ratio, both PG and TG act on the protein Therefore, the protein can be modified. When the ratio of PG is larger than the said ratio, the effect of PG is exerted excessively compared with TG, and the effect by combined use is not exhibited. In addition, when small, the effect of PG is too weak, and the combined effect cannot be expected very much.

The amount of protein glutaminase and transglutaminase added to the protein is PG: TG = 0.2 to 3.0: 1, preferably 0.2 to 2.3: 1 in the NMR signal intensity ratio. Regardless of the time of addition of PG and TG, PG and TG act on the protein, so that the protein can be modified. When the ratio of PG is larger than the said ratio, the effect of PG is exerted excessively compared with TG, and the effect by combined use is not exhibited. In addition, when small, the effect of PG is too weak, and the combined effect cannot be expected very much.

The NMR signal intensity ratio of the present invention measures the NMR spectrum of the labeled protein after isotopically labeling the glutamine residues of the protein by applying TG or PG to the substrate protein in the presence of the isotopically labeled ammonium salt ( ¹⁵ N or ¹⁴ N). It can be calculated from the NMR signal intensity. NMR measurement method is not particularly limited, for example, when the detection of the ¹⁵ N-labeled glutamine residue is the, ¹ H and ¹⁵ N of a correlation spectrum, for example, measuring the HSQC spectrum. When two or more signals are obtained, the sum of the signal intensities is calculated as the signal intensity. In other words, the NMR signal intensity ratio is expressed by "sum of signal strength when PG is applied" ÷ "sum of signal strength when TG is applied". As a labeling compound, if it is an ammonium salt, it is good, and ammonium chloride, ammonium sulfate, etc. are mentioned widely. ¹⁵ N-labeled and used as long as the nitrogen of the ammonium salt of ¹⁵ N, ¹⁴ N-labeled so long as it is by using the nitrogen of the ammonium salt of ¹⁴ N. The labeling method is in an aqueous solvent in the range of about pH 3.0 to about pH 9.0, preferably in the range of about pH 4.0 to about pH 8.0, in the range of about 4 to about 65 ° C, more preferably from about 25 to about It is good to keep the protein and ammonium salt to be labeled in the range of 60 ° C. Although there is no restriction | limiting in particular in reaction time, It is good to set it as about 30 second-1 week, More preferably, about 1 minute-about 1 day. In this reaction, the concentration of ammonium salt is preferably about 10 times or more, preferably about 200 times or more, relative to the concentration of the protein to be labeled, and the concentration of the protein to be labeled is in the range of about 1 μM to about 40 mM. More preferably, the concentration of the ammonium salt is in the range of about 10 μM to about 10 M. On the other hand, since proteins contained in foods such as α-lactoglobulin, casein, soybean globulin, myosin and act myosin can be isotopically labeled, the NMR signal intensity ratio of the present invention can be measured in the overall food containing protein. have.

An example is shown in which a protein is reacted with an isotopically labeled ammonium chloride by the action of TG or PG. When α-lactalbumin (hereinafter abbreviated α-La) is used as substrate, substrate solution (10 mg / ml α-La, 200 mM ¹⁵ NH ₄ Cl, 5% D ₂ O / 20 mM Tris-HCl (pH 7.0)), and TG or PG is added so that the substrate-enzyme ratio is 1000: 1, and then incubated for 26.5 hours at 37 ° C. The solution after incubation is transferred to an NMR sample tube and subjected to ¹ H- ¹⁵ N HSQC measurement using an Avance 600 manufactured by Bruker. When the carboxamide nitrogen to ¹⁵ N-labeled, since the combination of two ¹ H to ¹⁵ N, the two signals are observed as a pair for the chemical shift of ¹⁵ N 1. "Sum of signal intensity when PG was acted" ÷ "Sum of signal intensity when TG was acted" is calculated, and it is set as NMR signal intensity ratio.

The protein of the present invention is not particularly limited, but may include milk protein, whey protein, soy protein, wheat gluten, muscle protein, plasma, collagen, gelatin and the like. In addition, a mixture of two or more such proteins may be sufficient. As a foodstuff which concerns on this invention, as long as it contains the thing which modified the above-mentioned protein by combined use of PG and TG, the kind of raw material and a processing state do not matter. For example, sterilized state, degreased state, diluted state, concentrated state, dried state, and the state which processed variously are also included here.

Next, the enzyme preparation of the present invention will be described. In the enzyme preparation for protein modification of the present invention, the blending ratio of transglutaminase and protein glutaminase, which are essential constituents, is PG: TG = 0.05-3: 1 in terms of activity ratio, preferably PG: TG = 0.05 to 2: 1, but in the weight ratio PG: TG = 0.01 to 0.7: 1, preferably PG: TG = 0.01 to 0.4: 1, more preferably PG: TG = 0.01 to 0.3: 1 In terms of the NMR signal intensity ratio, PG: TG = 0.2 to 3.0: 1, preferably PG: TG = 0.2 to 2.3: 1, the condition is good, lactose commonly used in this field other than PG, TG, Various optional ingredients such as sucrose, maltitol, sorbitol, dextrin, branched dextrin, cyclodextrin, starch, polysaccharide, gum, pectin, and the like can also be blended. Moreover, you may contain vegetable proteins, such as an animal protein, soybean protein, and wheat protein. In addition, physiologically acceptable inorganic salts such as sodium bicarbonate, sodium citrate, sodium phosphate, sodium chloride and potassium chloride may also be blended into the present enzyme preparations as necessary. In addition, seasonings, sugars, spices, coloring agents, coloring agents, organic salts such as ascorbic acid, salts thereof, emulsifiers, fats and oils and the like can also be appropriately blended.

This invention also includes the method of finding out the appropriate addition ratio of protein glutaminase and a transglutaminase easily. That is, as described above, PG and TG are respectively acted on the protein separately in the presence of an isotopically labeled ammonium salt, the isotopically labeled functional group of the glutamine residue of the protein is measured, and the NMR signal intensity is measured to determine the NMR signal intensity ratio. Protein Glutaminase: Transglutaminase = 0.2 to 3.0: 1 By revealing the conditions of preferably 0.2 to 2.3: 1, the appropriate addition ratio of PG and TG can be found. The appropriate range of NMR signal intensity ratios of PG and TG is protein glutaminase: transglutaminase = 0.2 to 3.0: 1, preferably 0.2 to 2.3: 1, regardless of the type of substrate protein, and NMR Since there is a correlation between the signal intensity ratio and the appropriate addition ratio (activation ratio, weight ratio) of PG and TG in each matrix protein, the trial and error of the appropriate addition ratio (activation ratio, weight ratio) of PG and TG is determined by many experiments. It can be found simply without repeating it.

Although an experiment example and an Example are given to the following and this invention is demonstrated in more detail, this invention is not limited at all by these Examples.

Experimental Example 1

α-lactalbumin (α-lactalbumin, hereinafter abbreviated α-La) [manufactured by Sigma] was used as a substrate. Substrate solution (10 mg / ml α-La, 200 mM ¹⁵ NH ₄ Cl, 5% D ₂ O / 20 mM Tris-HCl (pH 7.0)) was prepared, and TG (Ajinomoto `` Actiba '' was prepared so that the substrate-enzyme ratio was 1000: 1. The enzyme purified from TG) or PG (derived from Chryseobacterium genus described in Patent Document 1) was added and then incubated at 37 ° C. for 26.5 hours. The solution after incubation was transferred to an NMR sample tube, and ¹ H- ¹⁵ N HSQC measurement was performed using NMR (Avaance 600 manufactured by Bruker). ¹ as a result of the H- ¹⁵ N HSQC measurement after 26.5 hours is shown in Fig. When the carboxamide nitrogen to ¹⁵ N-labeled, since the combination of two ¹ H to ¹⁵ N, the two signals are observed as a pair for the chemical shift of ¹⁵ N 1. From FIG. 1, it turned out that some of the carboxyamide nitrogen of the glutamine residue which exists in (alpha) -La is ^15- N-labeled, and succeeded in showing ^15- N-labeled by PG. Thus, by this method, it was shown that like TG, PG can react not only a deamide reaction but ammonium chloride with a glutamine residue. On the other hand, NMR signal intensity is the total sum of the area areas of the elliptic spots in FIG.

Example 1

To commercial milk, 0.2M ¹⁵ NH ₄ Cl, 5% D ₂ O, TG or PG described in Experimental Example 1 was added alone, respectively, and the signal intensity at each concentration was calculated. TG was added so that the substrate / enzyme ratio (S / E ratio) was 7400/1 by weight. In addition, PG was added 0.002-5 times of TG by weight ratio. The inactivation of TG used was 26 units per mg of enzyme protein, and the inactivation of PG was 120 units per mg of enzyme protein. The solution after the enzyme addition was incubated at 37 ° C. for 3 hours, then transferred to an NMR sample tube and subjected to ¹ H- ¹⁵ N HSQC measurement using NMR described in Experimental Example 1. Based on the amount of TG added, the amount of PG added thereto was expressed by the respective enzyme weight ratio (PG / TG) and activity ratio (PG / TG). And the result of having calculated | required PG / TG signal intensity ratio corresponding to each ratio is shown in Table 1.

Yogurt was prepared using commercial milk. 400 g of commercial milk was warmed to 50 ° C., and the amounts of TG and PG described in Experimental Example 1 described in Table 2 were added. The inactivation of TG used was 26 units per mg of enzyme protein, and the inactivation of PG was 120 units. After enzymatic reaction was performed at 50 ° C. for 90 minutes, the mixture was heated while mixing in a boiling bath. Upon reaching 90 ° C., it was immediately immersed in iced water and cooled to 45 ° C. 20 g (large raw material 5%) of a starter (Bulgaria yoghurt manufactured by Meiji New Kyo Co., Ltd.) was added, mixed well and dispensed into a tasting cup by 40 g. The lid was covered with aluminum foil and fermented at 38 ° C. for about 3-4 hours until the pH dropped to 4.5. It stored at low temperature (4 degreeC), and performed the physical property evaluation and the sensory evaluation the next day.

Physical properties measured the break stress using a texture analyzer (manufactured by Eiko Seiki). A 10 mm diameter columnar flanger was used and the breaking speed was 6 cm / min (1 mm / sec). The results are shown in Table 3. The breaking stress of the comparative product T added with only TG was significantly increased compared to the control. This is an effect by the crosslinking of the milk protein by TG. As for the inventions 1 to 4, too, the break stress was larger than the control, and the effect of increasing the break stress by TG was not impaired. However, since the break stress of the comparative product T-P was almost unchanged from that of the comparative product P, the TG reaction was almost stopped by PG, and it was confirmed that the effect of TG was hardly exhibited.

Six panelists evaluated the rigidity by sensory evaluation. When the reference product was scored as 0, the score was scored to be +5 for harder and -5 for softer, and the average value was obtained. The results are shown in Table 4. Compared with the control, the comparative product T added with only TG markedly increased rigidity. Also in the inventions 1-4, the increase of rigidity by TG was maintained. However, the rigidity of the comparative product T-P softens the yogurt like the comparative product P, which means that the TG reaction is almost stopped by PG, and the effect of TG is hardly exhibited. This result reflects approximately the result of the physical property measurement shown above. As a result of evaluating the preference of the overall texture, the present inventions 1 to 4 obtained a favorable evaluation result that the smoothness was improved compared to the case where only TG was added while maintaining the effect of improving the firmness of yogurt by TG. That is, the combined effect of PG and TG was confirmed.

Example 2

In order to adjust milk protein to 3.6% in Takanashi low fat milk (3.3% milk milk, 1.0% milk fat), 0.85% of skim milk powder (low heat type, containing 35% milk protein, manufactured by Yotsuban Kyo) is added, and heated at 55 ° C. It melt | dissolved and prepared the raw material oil for yoghurt. The crude oil was heated in a boiling bath, maintained at 95 ° C. for 2 minutes, and immediately cooled in an ice bath. When it became 47 degreeC, 0.006% of lactic acid bacteria starters (Yo-Flex, DVS YC-370, Christian Hansen) were added, and TG and PG were added as described in Table 5, and it stirred well. An aliquot was dispensed into plastic cups and fermented in an incubator at 44 ° C. until the pH reached 4.5-4.6. From the start of fermentation to the end, it generally required 4 to 5 hours, and after the end of fermentation, it was stored in a refrigerator (5 ° C). The following day, the surface completion of the obtained set type yoghurt was observed. In addition, the physical properties of the yogurt were evaluated by a texture analyzer (device maker: Stable Macro System, LTD.). Sensory evaluation was conducted with five trained panels. The measurement conditions of the physical properties of the yogurt were evaluated by a test speed of 1 mm / s, a flat flanged flanger having a diameter of 10 mm, and a breaking stress and adhesion area at 10% compression. The present inventors have already confirmed that the breaking stress has a high correlation with the creaminess of the yoghurt, and the firmness of the yoghurt. The results of the evaluation are summarized in Table 6.

The number of surface finishes was many in the control and the comparative 2, and a little more was observed in the comparative 3. In Comparative Products 1 and 1 and 2 of the present invention, no surface finish was found and the surface had a glossy surface. On the other hand, according to the physical property measurement and the sensory evaluation result, the comparative product 1 had a hard, soft agar-like texture, and when it was in the mouth, it felt moist. Comparative products 2 and 3 were harder than the control and the adhesion increased. In the sensory evaluation, there was a comment that it was smooth but was soft and lacked a sense of body. On the other hand, in the present inventions 1 and 2, both firmness and adhesiveness increased, there was a body feeling, and it was a smooth and creamy yogurt. As mentioned above, when it evaluated comprehensively from an external appearance, a physical property, and texture, it was shown that this invention 1 and 2 are the most preferable yoghurt, and the texture improvement of a set type low fat yoghurt is possible by using TG and PG in an appropriate balance.

Example 3

In order to adjust milk protein to 3.94% in Takanashi low-fat milk (3.3% milk milk, 1.0% milk fat), skim milk powder (low heat type, containing 35% milk protein, manufactured by Yotsuban Kyo) is added and dissolved while heating at 55 ° C. And raw material oil for yoghurt was prepared. The crude oil was heated in a boiling bath, maintained at 95 ° C. for 2 minutes, and immediately cooled in an ice bath. When it became 47 degreeC, 0.060% of lactic acid bacteria starters (Yo-Flex, DVS YC-370, Christian Hansen) were added, TG and PG were added as described in Table 7, and it stirred well. Aliquots were dispensed in stainless cups and fermented in an incubator at 44 ° C. until the pH reached 4.5-4.6. After fermentation was completed, the resultant was cooled in a refrigerator (5 ° C.), and then filtered through a mesh (216 μm) sieve to form a stud. The finished stud yoghurt was preserve | saved at 5 degreeC, and it was supposed to analyze the change of an external appearance and a texture in a storage step. In addition, the physical properties of yogurt were evaluated by a dynamic viscoelasticity measuring device (HAAKE, Leostress RS1). Sensory evaluation was conducted by three trained panels. The physical properties of yogurt were measured using a corn plate having a diameter of 6 cm, increasing the shear rate 0 → 100 [1 / s] for 300 seconds, and then immediately increasing the shear rate at 100 → 0 [1 / s] at the same time. A program to reduce over time was written. The measurement temperature was 10 degreeC and the viscosity at the shear rate of 100 [1 / s] was recorded. The results are summarized in Table 8.

The surface completion before studization was confirmed in a large amount in the control product and a small amount in the comparative product 2, but was hardly seen in the comparative product 1 and the inventions 1 to 4. As a result of the sensory evaluation immediately after studding, the control had a nod, a moist and soft texture. The comparative product 1 was provided with a body texture, but in the comparative product 2, the surface was shiny and smooth in the comparative product 2, but it was a moist and soft texture. All of the inventions 1 to 4 had surface gloss, and smoothness was also imparted with the body feeling. The measurement result of the viscosity is also in good agreement with the sensory evaluation, and the viscosity of the present inventions 1 to 4 was clearly higher than that of the control product or the comparative product 2, to support the imparting a body feeling. As a result of confirming the change in appearance and texture after refrigeration for 3 weeks, the comparison product 1 showed a lump, and the texture was remarkable, such as gritty sand, but the tendency was reduced in the invention 1, and in the inventions 2 to 4 Almost improved. When the above results were comprehensively evaluated, the present inventions 1 to 4 were obviously superior in terms of physical properties and texture (change in storage) as compared with the control products and the comparative products 1 and 2.

As described above, although yog (Comparative Product 1) using only TG has an effect of imparting a body feeling compared to no additives, quality deterioration during storage becomes a problem, and Yoghurt (Comparative Product 2) using only PG is smooth. The problem is moist texture without body. However, the yoghurt using the TG and PG in a constant addition ratio (Invention 1 to 4) not only solved this problem, but also appeared to be able to improve the quality by imparting surface gloss and smoothness.

Example 4

Commercial soy milk (Tai Shi unadjusted; final protein content 4.8%), 0.2M ¹⁵ NH ₄ Cl, 5% D ₂ O, TG or PG described in Experimental Example 1 were added alone and mixed well. TG was added so that the substrate / enzyme ratio (S / E ratio) was 6000/1 by weight. On the other hand, PG was added 0.01 to 1 times of TG. The solution after incubation at 37 ° C. for 1 hour and 15 minutes was transferred to an NMR sample tube, and ¹ H- ¹⁵ N HSQC measurement was performed using NMR described in Experimental Example 1. As in Example 1, Table 9 shows the results of calculating the PG / TG signal intensity ratios for each enzyme weight ratio (PG / TG) and activity ratio (PG / TG).

Tofu using TG and PG in combination at the addition ratios shown in Table 10 was prepared. In a platter container, 1 g of the commercially available soymilk was weighed in 10 g (corresponding to 3 g of brine) and a stainless steel jug in a 30% brine solution (manufactured by Akogasei Co., Ltd.). The enzyme solution was added to the soy milk, stirred and mixed, quickly poured into the assorted container containing the brine, and the stirring plate was moved up and down four times. The lid was covered and transferred to a 55 ° C. incubator and allowed to stand for 50 minutes. The tofu was transferred to a bat filled with water, left for 30 minutes to 1 hour, and then the tofu was cut out, transferred to a tofu container, and the contents were weighed. Water was added to the vessel until it was full, and the film was covered and heated to seal. It poured into the constant temperature water bath of 85 degreeC, heated for 45 minutes (secondary heating), and after that, the heat which remained with running water was removed, it was cooled in ice water, and refrigerated was preserved. The next day, the weight of the solid except for the water in the container was measured and used for physical properties and sensory evaluation.

The physical property evaluation performed the breaking test by the rheometer (manufactured by Fudogo Kogyo Co., Ltd.). The used flanger was made into 5 mm diameter disks, and breaking speed 6 cm / min (1 mm / sec). The sensory evaluation was performed by five panelists, with no enzyme added as 0 points, and 7 steps of evaluation were performed between ± 3 points for smoothness and hardness, and the average value was obtained. Table 11 lists the results. The comparative stresses T1 and T2 added with TG increased the break stress in comparison with the control. On the other hand, the comparative stresses P1 and P2 to which PG was added reduced the break stress compared to the control. Invention 1 of the present invention 1 was almost equivalent to comparative stress T1 to which the same amount of TG was added, and taste changed more smoothly. Although the breakdown stress of the invention 2 slightly decreased compared with the comparative product T2 to which the same amount of TG was added, the breakage stress was still larger and smoother than that of the control product. In the invention 3, the break stress was reduced compared to the comparative product T2 to which the same amount of TG was added, but the break stress was larger than the comparative product P2 to which the same amount of PG was added, and the effect of adding TG was confirmed. The comparative product T-P was soft to the same extent as the comparative products P1 and P2, and had already exhibited a hardening effect even though it was based on the TG reaction. This means that the TG reaction is almost stopped by PG. In addition, as a result of the sensory evaluation, in any of the present inventions 1 to 3, the hardening by TG was maintained, the taste was improved compared to the addition of TG alone, and the overall preferred texture was imparted. That is, the combined effect of PG and TG was confirmed.

Example 5

Udon and mid-screen were prepared in the mixing and starting process shown in Table 12. The transglutaminase and the protein glutaminase were dissolved in water at the time of hydrolysis. Table 13 shows the amount of each enzyme added to the udon noodles and the middle noodles. Udon noodles were boiled for 20 minutes with 15 times the amount of boiled water for 100 g of cotton, and evaluated by the panel after which the texture was trained (n = 5). The Chinese noodles were evaluated like udon except that the boiling time was 5 minutes. Based on the reference product, the items of hardness, viscosity, and smoothness were evaluated (obviously weakened: ××, slightly weakened: ×, equivalent: Δ, somewhat stronger: ○, apparently stronger: ◎). Since the evaluation results of udon and mid-screen were almost the same tendency, it was summarized in Table 14 and described. When the PG / TG ratio was 0.01, 0.05, or 0.2 (inventions 1, 2, and 3, respectively) in the weight ratio in all of the udon and mid-sized noodles, the texture was balanced with the firmness, viscosity, and smoothness of the noodles. . That is, it was also shown in the example of noodles that it is a suitable ratio which acts both transglutaminase and protein glutaminase, and acquires the preferable effect of both.

According to the present invention, a protein modification effect different from the case where each enzyme of transglutaminase and protein glutaminase is used alone can be obtained, and protein foods having completely novel characteristics can be produced. In addition, appropriate conditions under which both TG and PG enzymes act on the substrate can be easily identified. Therefore, the present invention is very useful in the food field.

In addition, each indication, such as said patent document, shall be referred to and introduce | transduced into this book. Within the scope of the entire disclosure (including the scope of the claims) of the present invention, and on the basis of the basic technical idea, it is possible to change or adjust the embodiments or examples. In addition, various combinations and selections of various disclosure elements are possible within the framework of the claims of the present invention. That is, the present invention obviously includes various modifications and modifications that can be made by those skilled in the art according to the entire disclosure and technical spirit including the claims.

Claims (10)

In the method of modifying a protein by applying protein glutaminase and transglutaminase to a protein, the addition time of the protein glutaminase to the protein is substantially the same as the addition time of the transglutaminase or Or a method of modifying a protein before transglutaminase acts on the protein. The method according to claim 1, wherein the amount of protein glutaminase and transglutaminase added to the protein is protein glutaminase: transglutaminase = 0.05 to 3: 1 in activity ratio. The amount of the protein glutaminase and the transglutaminase to be added to the protein in terms of weight ratio, wherein the protein glutaminase: transglutaminase = 0.01 to 0.7: 1 How to be. The protein glutaminase: transglutamina according to any one of claims 1 to 3, wherein the amount of the protein glutaminase and the transglutaminase added to the protein is in the NMR signal intensity ratio. The modification method is a condition which becomes == 0.2-3.0: 1. 5. The method of claim 1, wherein the protein is one or more selected from milk protein, whey protein, soy protein, wheat gluten, plasma, muscle protein, collagen and gelatin. Food containing a protein modified by the method according to any one of claims 1 to 5. Transglutaminase: Protein glutaminase The ratio of protein glutaminase: transglutaminase = 0.05-3: 1 in the activity ratio of the enzyme formulation for protein modification. Transglutaminase: Protein glutaminase The ratio of protein glutaminase: protein glutaminase: transglutaminase = 0.01 to 0.7: 1 enzyme preparation for protein modification. The protein modification characterized in that the compounding ratio of transglutaminase: protein glutaminase is such that protein glutaminase: transglutaminase = 0.2 to 3.0: 1 in the NMR signal intensity ratio Enzyme preparation. The transglutaminase and the protein glutaminase were acted on the protein separately in the presence of an isotope-labeled ammonium salt, and the isotope-labeled functional group of the glutamine residue of the protein was measured, and the NMR signal intensity was measured. A method for discovering the proper addition ratio of protein glutaminase and transglutaminase.

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